1) Field of the Invention
The present invention relates to miRNA triplex formations capable of downregulating viral replication. More particularly, the present invention relates to the discovery of miRNAs having high homology with both HIV-1 and a co-infecting virus such as HHV-6, HHV-7, or GVB-C, and the use of such mutually homologous miRNAs to form stable triplex molecules effective in the downregulation and/or inhibition of viral replication.
2) Description of Related Art
The general concept of harnessing RNA interference to silence gene replication/transcription/expression for therapeutic benefit is known in the art, as is the idea of using single-stranded interfering RNA molecules (e.g., antisense RNA, small interfering RNA (siRNA), microRNAs (miRNA)) to silence expression of viral genes, for example, as part of a component in an antiviral therapy. Further efforts in the prior art to control viral replication include the use of attenuated viral mutants or viral sequences, subunits or particles that can be used to generate a protective immune response against live virus (i.e., viral particles that are sufficiently “virus” looking to stimulate host immune system to generate protective antibodies but sufficiently disabled or incomplete to prevent onset of negative viral effects). Also, considerable attention has been directed towards the use of proteins, peptides or chemicals having therapeutic antiviral potential that operate by inhibiting an essential viral activity in vivo, acting on the virus itself to forestall/inhibit/prevent its negative consequences. None of these efforts involve the use of stable triplex formations to inhibit viral replication; rather, they utilize completely different mechanisms. Further, the prior art is replete with other efforts directed towards the use of viral sequences having utility in the context of the construction of viral vectors for the delivery of genes or proteins of interest (i.e., in the context of gene therapy) or in the context of a vaccine adjuvant.
For example, U.S. Pat. No. 6,682,907 describes the use of triplex structure DNA in transferring nucleotide sequences. The structure of the DNA triplex formed during reverse transcription in this process enables, or at least contributes, to the entry of the retroviral genome into the cell nucleus, thus allowing infection of non mitotic cells. The invention concerns a nucleotide sequence of retroviral or retroviral-like origin, which can be prepared synthetically, comprising cPPT and CTS regions which are cis-acting in reverse transcription in general, and in particular two associated polynucleotides when they are placed in the normal retroviral genome, each polynucleotide containing at least 10 nucleotides. The vector of the invention contains a transgene inserted under the control of viral or non viral sequences regulating transcription or expression. Ultimately, the triplex formation with HIV in this invention is intended to promote an immune response in the body, and does not disclose specific sequences that effect HIV replication by forming stable triplexes in vivo.
U.S. Pat. No. 6,555,342 describes a fusion protein delivery system and uses thereof. Ultimately, this invention demonstrated that Vpr and Vpx can serve as vehicles to deliver functionally active enzymes to the HIV virion, including those that may exert an antiviral activity such as SN. However, there is no discussion of using the specific miRNA or siRNA sequences identified in the present invention to form stable triplexes to inhibit HIV replication.
U.S. Pat. No. 6,531,123 describes lentiviral vectors and contemplates attenuated lentiviruses, and improved viral packaging and transducing vectors derived from lentiviruses, especially HIV-1, useful for the delivery of nonlentiviral genes to target cells. It also contemplates the use of these vectors in delivering transgenes to target cells, especially nondividing cells, in organisms, especially humans. However, there is no discussion of using the specific miRNA or siRNA sequences identified in the present invention to form stable triplexes to inhibit HIV replication.
U.S. Patent No. 20070099858 describes small nucleic acid molecules, such as short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules capable of mediating or that mediate RNA interference (RNAi) against influenza virus gene expression. However, there is no disclosure of using miRNA to form triplex formation to inhibit the HIV.
U.S. Patent No. 20030175693 describes an HIV recombinant vaccine. The invention encompasses recombinant HIV and SIV viruses containing heterologous transcriptional regulatory elements in the U3 region of the virus. In particular embodiments, the recombinant virus has decreased replication in vivo and the virus has a protective effect when administered to a host. The recombinant virus can have heterologous transcriptional regulatory elements that replace the HIV region corresponding to the NFKB/Sp1/TATA Box/initiation region (−114 to +1) or corresponding to the NFKB/Sp1/TAR region (−114 to +93) of the SIVmac239 long terminal repeat. However, there is no disclosure of using miRNA to form triplex formation to inhibit the HIV.
In each instance, the prior art fails to teach the use of specific miRNA sequences such as those identified in the instant application to form stable triplexes with HIV to inhibit HIV replication in accordance with the teachings of the present invention. Thus, although the present invention arises in view of the noted background, it nevertheless diverges therefrom in the discovery of specific miRNA sequences capable of forming with the HIV-1 genome stable triplexes that inhibit HIV-1 replication in vivo. These miRNA sequences find utility in the context of pharmaceutical compositions for use in the treatment and prevention of the HIV, and given the conserved nature of many viral genomes, may be reasonably extended to the treatment of other viruses, particularly retroviruses, more particularly lentiviruses such as SIV etc.
Accordingly, it is an object of the present invention to provide mutually homologous miRNAs give rise to the creation of stable triplex formations with a viral genome, such as the HIV-1 genome, to effect the downregulation of viral replication in vivo.